N34S mutation in the SPINK1 gene is not associated with alternative splicing

Previous studies have shown an association between chronic pancreatitis (CP) and mutations, especially the N34S mutation, in the serine protease inhibitor Kazal type 1 (SPINK1) gene. But the underlying molecular mechanisms are unknown. The aberrant splicing caused by the cosegregating intronic mutations might play a role, but this hypothesis has not been tested. We here examined the messenger RNA sequences of the SPINK1 gene in patients carrying the mutations.

RNA was isolated from the surgically resected pancreas of 2 CP patients carrying the homozygous N34S mutation and from the gastric biopsy specimen of a CP patient carrying the heterozygous [-215G>A; IVS3+2T>C] mutation. The entire coding region of the SPINK1 gene was amplified by reverse transcription-polymerase chain reaction, subcloned, and sequenced. The level of the wild-type SPINK1 transcript was assessed by real-time polymerase chain reaction.

Alternative splicing was not associated with the N34S mutation. On the other hand, the [-215G>A; IVS3+2T>C] mutation caused skipping of whole exon 3, where the trypsin binding site is located. This mutated protein was predicted to consist of 63 amino acids: deletion of amino acid sequence from residues 30 to 64 and shifting of reading frame at amino acid 65 with a novel stop codon. The expression of the wild-type SPINK1 transcript was decreased to 62% of the healthy control in the CP patient carrying the heterozygous [-215G>A; IVS3+2T>C] mutation.

Splicing mutation might represent a mechanism for SPINK1-associated CP, but the N34S mutation is not associated with alternative splicing.